Cell migration is critical in many physiological processes. Chemotaxis, for example, is the study of cell motion in response to a soluble chemo-attractant stimulus. Similar and related mechanisms include haptotaxis and chemoinvasion, which rely on cell motility on a substrate-bound stimulus and movement through an Extracellular Matrix (ECM) boundary layer, respectively. These processes play a vital role in the study of many therapeutic areas including oncology, inflammation and angiogenesis. In recent years there have been many advances in the understanding of this physiological response, and much work has been done on the various classes of cytokines (e.g. TNFa, IL-1) and chemokine receptors (e.g. CCR2, CCR5).
The most common technique for measuring cell migration in vitro is via a measurement geometry known as the Boyden chamber [1] first described in 1962. This geometry, developed by Dr. Stephen Boyden consists of two chambers separated by a porous membrane as depicted in FIG. 1. When using a Boyden chamber as a chemotaxis measuring device, a chemoattractant is typically added to the lower chamber, cells are added to the upper chamber and the porous membrane serves as a means to establish a diffusion-based chemical gradient between the upper and lower chambers. The cells on the top side of the membrane detect this gradient, migrate to the individual pores in the membrane, and then crawl through the holes to the lower chamber. Once migrating through the pore, the cells ultimately either fall through the membrane to a lower reservoir, or end up migrating on to the bottom side of the membrane. Determination of the chemotactic response relies on quantifying the number of cells that migrated through the membrane in relation to the total number of cells added to the top chamber.
There have been many small improvements to the standard Boyden chamber geometry since its inception, but the basic device geometry and membrane components have remained fairly consistent. Currently, there are many commercial sources for 6-well, 24-well or 96-well. Boyden chamber-derived chemotaxis kits, including the ChemoTx™ system sold by Neuroprobe Inc. (Gaithersburg, Md.), the Transwell™ system sold by Corning Life Science (Acton, Mass.) and the HTS Fluoroblok™ system sold by Becton-Dickinson (Franklin Lakes, N.J.). All of these devices are basically rectangular arrays of Boyden chambers using microplate formats and injection mold fabrication techniques for the upper and lower reservoirs. All of these commercial devices also use the same basic material for the porous membrane which is known as a “Track-Etch Membrane”.
Track-etch membranes are manufactured by exposing thin polymer films (e.g. polyester, or polycarbonate) to radioactive particle bombardment, followed by chemical etching [2]. The results of this manufacturing process are a thin film with a random pattern of very defined micro-holes as shown in the brightfield image of FIG. 2. The density of micro-holes using this fabrication technique is governed by the exposure time and exposure geometry in relation to the radioactive source. The size of the micro-holes in a given track-etch membrane is controlled by a combination of time, temperature and the chemical concentration used during the etch step. Typical etch solutions include highly concentrated NaOH, or HF. The micro-hole size is very uniform, and in general fairly orthogonal to the surface of the membrane. Pore size typically ranges from 0.2 microns, up to 10 microns in diameter. For filtering applications, it is generally better to have higher pore densities. However, due to the random nature of the ionization particle bombardment, pore density has a practical upper limit so as to avoid the random occurrence of “doublets”, i.e. two pores touching each other. As such the “porosity” of track-etched membranes, defined as the area of pores divided by the area of non-pore, is generally on the order of a few percent.
The predominant application of such membranes is for fine particle and contaminant filtering of fluids as well a method of capturing and detecting microorganisms. The filtering applications take advantage of the very uniform and defined pore size of the membrane. This characteristic makes these types of membranes ideal for precisely filtering particles or micro-organisms of a given size. For biological applications such as cell migration, track-etch membranes of 2, 3, 5 and 8 micron diameter pore sizes are most commonly used. For these applications, pore size is often matched to size of the cells being studied, bigger cells use bigger pores. Typically, the pore size is chosen to be slightly larger than the nucleus of the cells being studied. Most of the commercial manufacturers of Boyden-style chemotaxis chambers offer a variety of products incorporating different pore sizes. They also offer membranes in different materials, most commonly polycarbonate and polyester, and supply biological substrate coatings (e.g. collagen, fibronectin or laminin) or surface coating protocols. These biological coatings are sometimes useful so as to more accurately mimic the in vivo surface/adhesion biology of migrating cells.
The quantitative read-out from a Boyden chamber chemotaxis assay is based on a count of the number of cells which migrate through the membrane towards the chemoattractant in the lower chamber. Quite often, one must also employ negative control measurements where no chemoattractant is used in the lower chamber to correct for random migration effects. In existing commercial Boyden chamber technologies, quantification of the number of cells is accomplished by using fluorescent dye labeling of the cells. Labeling of the cells is necessary as the cells cannot be visualized on the surface of the track-etch membranes directly without using a fluorescent marker. Once labeled, the cells can be counted directly using cell counting microscopy; or if a proportionality relationship can be established between fluorescence and cell number, a bulk fluorescent measurement can be made which is proportional to cell number. Cells are typically labeled at the beginning of the experiment, i.e. before cell migration occurs. Cells can also be “post-labeled”, i.e. after the cell migration occurs. This latter method is often preferred when working with time-sensitive or label-sensitive cell types.
Boyden chamber technology is the current “gold standard” for in vitro chemotaxis assays and has been around for almost fifty years. The modern incarnations of the technique have the advantage of being amenable being amenable to multi-well microplate formats and the precision of plastic injection molding techniques; as such they are fairly high throughput and reasonably priced. While being the current gold standard, and clearly dominating the research market, there are several disadvantages to the current Boyden chamber systems. These disadvantages will be discussed in the following paragraphs